1. Field of the Invention
The present invention relates generally to the fields of biology and medicine. In particular, the invention relates to methods of producing collagen IV.
2. Description of Related Art
Collagen IV scaffolds are critical components of basement membranes (BM), a specialized form of extracellular matrix that underlies all epithelia in metazoa from sponge to human. Collagen IV molecules are assembled into networks that support the assemblage of BM components (Hudson et al., 2003). The scaffolds confer structural integrity to tissues, provide a foundation for the assembly of other macromolecular components, and serve as ligands for integrin cell-surface receptors that mediate cell adhesion, migration, growth and differentiation (Moser et al., 2009; Hynes, 2002; Yurchenco and Furthmayr, 1984). The networks also participate in signaling events in Drosophila development, in the clustering of receptors in the development of mammalian neuromuscular junction (Fox et al., 2007), and they are involved in autoimmune and genetic diseases (Gould et al., 2006; Gould et al., 2005; Hudson et al., 2003).
The collagen IV networks are assembled by oligomerization of triple-helical protomers by end-to-end associations and by intertwining of triple helices through their N- and C-terminal domains (Khoshnoodi et al., 2008; Khoshnoodi et al., 2006). At the C-terminus, two protomers associate through their trimeric non-collagenous (NC1) domains forming a hexamer structure. The protomer-protomer interface is covalently crosslinked, a key reinforcement that strengthens the structural integrity of networks. In the case of humans, the crosslink also confers immune privilege to the collagen IV antigen of Goodpasture autoimmune disease (Vanacore et al., 2008; Borza et al., 2005).
The inventors previously identified a sulfilimine bond (Met93-S═N-Hyl211) that stabilizes the NC1 trimer-NC1 trimer interaction in which the sulfur atom of methionine-93 (Met93) residue from one NC1 domain connects to the ε-nitrogen atom of hydroxylysine-211 (Hyl211) of an interacting NC1 domain (Vanacore et al., 2009). The crystal structure of the NC1 hexamer (Sundaramoorthy et al., 2002) demonstrates that this bond plays a critical role in not only stabilizing the quaternary structure of the NC1 hexameric complex but also as reinforcement to the entire collagen IV network. The inventors have also determined that the enzyme responsible for this bond formation is human peroxidasin (PXDN).
The sulfilimine bond likely occurs in diverse metazoan species. NC1 dimer subunits, a signature structural feature indicative of crosslinks, have been identified in collagenase digests of basement membranes including human (Weber et al., 1984), bovine (Weber et al., 1984), dog (Thorner et al., 1996), and mouse (Weber et al., 1984). Furthermore, a phylogenetic analysis of the Lys211 and Met93 residues, based on a multiple sequence alignment of the NC1 domain across the metazoan phylum (Vanacore et al., 2009; Aouacheria et al., 2006), revealed that the sulfilimine bond may occur in many metazoans, except in hydra, flatworm, sponge, and placozoa. A further comparison of the sequence motif (X-K-A/S/G) that confers hydroxylation of lysyl residues by lysyl hydroxylase (Kivirikko and Pihlajaniemi, 1998) occurs in the NC1 domains of all metazoa except hydra, sponge and placozoa. The motif is also absent in the α4 NC1 domain of human, mouse, bovine and chick, which in the case of bovine Lys211 does not undergo hydroxylation and leads to the formation of s-lysyl-methionine crosslink (Vanacore et al., 2008). Phylogenetic analysis suggest that the sulfilimine crosslink is a key biologic feature for tissue organization, development, and maintenance.
Though a great deal has been learned about collagen IV formation and crosslinking, including key enzymes in its regulation, further insights into how to exploit this molecule and the machinery that forms it in therapy remain to be uncovered.